Polypeptides that mimic isll and uses thereof

Abstract

This invention discloses an Isl1-mimicking polypeptide, TAT-Isl1-ELE-1, containing 40 amino acids and consisting of a TAT sequence that promotes polypeptide entry into cells, a linker peptide, and a target sequence. The target sequence is amino acids 121-141 of the human Isl1 protein. The TAT-Isl1-ELE-1 polypeptide possesses Isl1-like functions and can effectively reprogram mouse spinal cord reactive astrocytes into motor neuron-like cells. Based on the amino acid sequence and spatial structure of this polypeptide, a novel small molecule drug can be designed, which can be synthesized in large quantities and is easy to operate clinically. This provides a new and viable approach for cell replacement therapy and regenerative medicine research after spinal cord injury (SCI), thereby achieving better SCI repair and functional reconstruction effects.

Description

Technical Field

[0001] This invention relates to a polypeptide and its applications, and more particularly to a polypeptide that mimics Isl1 and its applications. Background Technology

[0002] The primary function of motor neurons is to innervate skeletal muscles and control their movement. As a special type of neuron, motor neurons are mainly distributed in the spinal cord, and their degeneration or loss is a characteristic manifestation of spinal cord injury (SCI). Previous studies have shown that human fibroblasts can be reprogrammed into motor neurons by regulating the expression of organic cation / carnitine transporter 4 and LIM homeobox 3 (Lhx3) in vitro. Furthermore, transplanting these reprogrammed motor neurons into the injured spinal cord of SCI-inducing rats has shown a significant effect in promoting motor function recovery. The inventors' previous research found that in an SCI mouse model, PTB knockdown successfully replenished motor neuron-like cells in the injured spinal cord, thereby promoting motor function recovery in mice. These findings highlight the important therapeutic role of motor neuron replenishment in promoting functional recovery after SCI. Given the crucial role of motor neurons in restoring motor function after SCI, exploring and developing additional strategies to effectively replenish these cells is essential.

[0003] Spinal motor neurons co-express Lhx3, Hb9, and Isl1 at birth, while branch motor neurons and visceral motor neurons in the central nervous system (including the ventral midbrain, hindbrain, and cervical spinal cord) co-express Isl1, Tbx20, and Phox2a / 2b. Both studies confirm a close association between Isl1 and motor neurons. Therefore, exploring whether the combined effects of Isl1 and other factors can effectively promote cell reprogramming is crucial. Recent research shows that Isl1 overexpression combined with Ngn2, Sox11, and Lhx3 can directly reprogram human fibroblasts into motor neurons. Mouse fibroblasts can also achieve motor neuron reprogramming through a combination of neuronal transcription factors (Ascl1, Myt1l, and Pou3f2) and motor neuron-specific transcription factors (Lhx3, Hb9, Isl1, and Ngn2). Furthermore, the combined addition of Neurod1 to Ascl1, myt1l, Pou3f2, Lhx3, Ngn2, Isl1, and Hb9 promotes the transdifferentiation of human fibroblasts into functional motor neurons. These induced motor neurons are excitable and capable of generating action potentials. Overexpression of transcription factors Isl1, Lhx3, and Ngn2 has been shown to transdifferentiate induced pluripotent stem cells (iPSCs) into motor neurons. These studies suggest that Isl1 plays a crucial role in the specialization process of motor neurons. Previous research by the inventors found that the combined application of Isl1 with three other classic transcription factors (Ascl1, Myt1l, and Pou3f2) successfully achieved in vitro reprogramming of reactive rat and human spinal cord astrocytes, causing them to differentiate into motor neuron-like cells, indicating that Isl1 has broad application prospects.

[0004] However, no peptide that mimics Isl1 has yet been shown to promote neuronal regeneration and functional recovery in the damaged spinal cord. Summary of the Invention

[0005] Purpose of the invention: The purpose of this invention is to provide a TAT-Isl1-ELE-1 that mimics the function of Isl1, and the application of this polypeptide in reprogramming spinal cord reactive astrocytes into motor neuron-like cells in vitro.

[0006] Technical solution: The present invention provides a polypeptide that mimics Isl1, wherein the polypeptide is TAT-Isl1-ELE-1 polypeptide, and its amino acid sequence is GRKKRRQRRRPQGGSGHSGALREDGLFCRADHDVVERASL.

[0007] To mimic the function of Isl1 and find a drug that can be synthesized in large quantities and is convenient to administer, the inventors first screened three target sequences from the conserved functional domains of Isl1 based on phylogenetic tree analysis. Further experimental verification revealed the TAT-Isl1-ELE-1 peptide, which can reprogram mouse spinal cord reactive astrocytes into motor neuron-like cells. The design of this peptide sequence and spatial structure will contribute to new drug development.

[0008] In the amino acid sequence, GRKKRRQRRRPQ is a TAT sequence that promotes the entry of the polypeptide into the cell, GGSGHSG is a linker peptide, and ALREDGLFCRADHDVVERASL is the target sequence, which is amino acid 121-141 of the human Isl1 protein (accession: P61371).

[0009] The present invention also provides a pharmaceutical composition comprising the polypeptide and a pharmaceutically acceptable carrier.

[0010] The carrier is preferably an excipient, suspending agent, filler, or diluent.

[0011] The preferred dosage form of the pharmaceutical composition is an injectable form.

[0012] The present invention also provides the use of the said polypeptide or the said pharmaceutical composition in inducing cell transformation.

[0013] Furthermore, the transformation results in a cellular morphology that shifts towards a neuron-like structure and is reprogrammed into a neuron.

[0014] Furthermore, the neurons are MAP2+ mature neurons and ChAT+ motor neurons. MAP2 and ChAT are neuronal biomarkers and motor neuron-specific biomarkers, respectively.

[0015] The cells are preferably reactive astrocytes, and most preferably spinal cord reactive astrocytes.

[0016] The present invention also provides the use of the peptide in the preparation of drugs for treating SCI.

[0017] Beneficial Effects: Compared with existing technologies, this invention has the following significant advantages: The TAT-Isl1-ELE-1 polypeptide provided by this invention has Isl1-like functions and can effectively reprogram mouse spinal cord reactive astrocytes into motor neuron-like cells. The novel small molecule drug designed based on the amino acid sequence and spatial structure of this polypeptide can be synthesized in large quantities and is easy to operate clinically, providing a new and selectable approach for cell replacement therapy and cell regenerative medicine research after spinal cord injury (SCI), thereby achieving better SCI repair and functional reconstruction effects. Attached Figure Description

[0018] Figure 1 Image of GFAP staining in primary mouse spinal cord astrocytes;

[0019] Figure 2 The mRNA levels of GFAP in spinal cord astrocytes of mice treated with LPS for 24 h were compared between the control group and the control group.

[0020] Figure 3 Light micrographs of reactive astrocytes in the spinal cord of mice treated with the control group and three TAT-Isl1-ELE peptides;

[0021] Figure 4 Fluorescence images of neurons and motor neurons in the spinal cord reactive astrocytes of TAT-Isl1-ELE-1 reprogrammed mice. Detailed Implementation

[0022] The technical solution of the present invention will be further described below with reference to the accompanying drawings.

[0023] Example 1

[0024] This embodiment uses phylogenetic tree analysis to compare conserved functional domains in Isl1, screens out three target sequences, and designs three peptides that mimic Isl1: TAT-Isl1-ELE-1, TAT-Isl1-ELE-2, and TAT-Isl1-ELE-3.

[0025] The amino acid sequences of these three polypeptides correspond to SEQ ID NO.1-3, respectively. They are all composed of a TAT sequence (GRKKRRQRRRPQ), a linker peptide (GGSGHSG), and a target sequence. The three target sequences correspond to amino acids 121-141, 1-10, and 301-310 of the human Isl1 protein, respectively.

[0026] Example 2

[0027] This embodiment verifies the effect of the three peptides in Example 1 on the reprogramming of mouse spinal cord reactive astrocytes.

[0028] Experimental materials: TAT-Isl1-ELE-1, TAT-Isl1-ELE-2, TAT-Isl1-ELE-3 (Qiangyo Biotechnology), primary mouse spinal cord astrocytes, DMEM-F12 (Thermo), FBS (Gibco), LPS (Sigma-Aldrich), Neurobasal (Gibco), sodium selenite (Sigma-Aldrich), insulin (AbMole), putrescine (APExBIO), progesterone (Sigma-Aldrich), B-27TM (Gibco), SB431542 (Sigma-Aldrich), ChIR99021 (Sigma-Aldrich), db-cAMP (APExBIO), NT-3 (Peprotech), CNTF (Peprotech), GDNF (Peprotech), BDNF (Peprotech).

[0029] The experimental method consists of four parts, as detailed below.

[0030] 1. Preparation of TAT-Isl1-ELE-1, TAT-Isl1-ELE-2, and TAT-Isl1-ELE-3 polypeptide solutions.

[0031] The lyophilized peptide powder was dissolved in sterile water and stored at a concentration of 1 mg / mL. It was dispensed in 100 μL containers and stored at -80℃.

[0032] 2. Primary culture yielded mouse spinal cord astrocytes with high purity.

[0033] Spinal cord tissue was obtained from newborn mice. The spinal cord was rinsed twice in a culture dish containing ice-cold D-Hanks solution. The surface tunica albuginea and blood vessels were carefully removed using microsurgical forceps. The spinal cord was rinsed 2-3 times with D-Hanks solution and transferred to another culture dish. The spinal cord was minced until it became chyme-like. The minced spinal cord pieces were transferred to 15 mL centrifuge tubes, and an equal volume of 0.25% trypsin was added. The mixture was incubated in a 37°C water bath for 15 min, with the mixture being pipetted and mixed every 5 min until no obvious tissue fragments remained. Twice the volume of 10% FBS complete medium was added to terminate the enzyme digestion. The mixture was centrifuged at 1000 rpm for 5 min, the supernatant was discarded, and the cell pellet was collected. The cells were resuspended in 5 mL of basal medium and centrifuged twice. The cells were resuspended in 10% FBS complete medium, and the cell suspension was evenly distributed by pipetting. The mixture was filtered through a 200-mesh sieve to prepare the initial cell suspension. The initial cell suspension was seeded into 25 cm⁻¹ cells. 2In a culture flask, incubate upside down for 20 min in a 37℃, 5% CO2 incubator. Then gently invert the flask, remove the cell suspension, and remove the fibroblast components. Centrifuge the cell suspension at 1000 rpm for 5 min, add it to 5 mL of complete culture medium containing 10% FBS, gently mix, and count the cells using a cell counter. Dilute the mixture with 10% FBS complete culture medium to a concentration of 5 × 10⁻⁶ cells / mL. 5 / mL, take 15 mL and inoculate at 75 cm 2 In culture flasks, the medium is completely replaced every two days to remove non-adherent dead cell debris, allowing glial cells to grow fully. Observe under a microscope each time the medium is changed. After about one week, the cells will cover the bottom of the flask, and then further purification and culture can be carried out. When the glial cells have been cultured for 7-9 days and have covered the bottom of the culture flask, place it on a constant-temperature shaking culture bed at 37°C and 280 rpm / min for 16-18 hours (the constant-temperature culture bed should be sterilized with UV light beforehand). After shaking, remove the culture flask and sterilize it with 75% alcohol. After removing the cell suspension (mainly oligodendrocytes and microglia), wash twice with fresh culture medium. The remaining bottom layer cells are mainly astrocytes. Add 1 mL of 0.25% trypsin for digestion, gently shake the culture flask back and forth and side to side. Observe under a microscope that the cell bodies have shrunk and become rounded. Immediately stop the digestion with 10% FBS complete medium, then gently scrape the cells off with a cell scraper. Collect the suspended cells into a 15 mL centrifuge tube using a sterile pipette, centrifuge at 1000 rpm / min for 5 min, repeat twice, and finally seed the cells in a 75 cm⁻¹ incubator. 2 Cultured in culture flasks. To ensure cell viability and purity, spinal astrocytes of passage 2-3 are generally selected for subsequent experimental studies.

[0034] 3. Construction of a primary mouse spinal cord reactive astrocyte model.

[0035] Second- to third-generation spinal astrocytes were selected and stimulated with 10 μg / mL LPS for 24 h. The expression of the astrocyte-specific gene GFAP was increased after LPS stimulation by real-time RT-PCR, which confirmed that the cells were activated into reactive astrocytes.

[0036] 4. Treatment of mouse spinal cord reactive astrocytes with TAT-Isl1-ELE peptide.

[0037] Three TAT-Isl1-ELE peptides were used to infect reactive astrocytes. After 2 days, the infection medium was replaced with induction medium (N3 / basal medium). The medium was then partially replaced every 3 days for 28 days. Immunocytochemistry was used to detect the expression of MAP2 panneuronal markers and ChAT motor neuron markers.

[0038] Specifically: Reactive astrocytes were resuspended in DMEM / F12 containing TAT-Isl1-ELE-1, TAT-Isl1-ELE-2, TAT-Isl1-ELE-3 peptides and 10% FBS. The cell and peptide mixture was seeded into cell culture plates (six wells), with each well containing four 12 mm poly-L-lysine-pretreated glass cell spreaders at a seeding density of 20,000 cells / well and a peptide concentration of 40 μg / mL per well. After two days, the cell culture medium was replaced with induction medium. The induction medium consisted of equal volumes of DMEM / F12 and Neurobasal medium containing 30 nM sodium selenite, 20 μg / mL insulin, 100 nM putrescine, 20 nM progesterone, 2% FBS, and 0.4% B-27. TM The induction medium consisted of 10 μM SB431542, 1 μM ChIR99021, 1 mM db-cAMP, 10 ng / mL GDNF, 10 ng / mL BDNF, 10 ng / mL CNTF, and 10 ng / mL NT-3. The medium was partially replaced every three days.

[0039] The experimental results consist of three parts, as detailed below.

[0040] 1. Primary mouse spinal cord astrocyte culture.

[0041] Primary cultures of neonatal mouse spinal cord astrocytes were used, and cell purity was determined by staining with the astrocyte marker GFAP. Figure 1 As shown, most of the cultured primary mouse spinal cord astrocytes were positive for GFAP staining.

[0042] 2. Construction of an in vitro mouse spinal cord reactive astrocyte model.

[0043] LPS stimulation of spinal cord astrocytes showed that LPS stimulation significantly promoted GFAP mRNA expression in the cells, such as... Figure 2As shown, after treating mouse spinal cord astrocytes with 10 μg / mL LPS for 24 h, the mRNA level of GFAP was significantly increased compared with the control group. This indicates that a spinal cord reactive astrocyte model has been successfully constructed and can be used for subsequent experimental studies.

[0044] 3. Treatment of mouse spinal cord reactive astrocytes with TAT-Isl1-ELE peptide.

[0045] After 28 days of treatment, cells treated with TAT-Isl1-ELE-1 peptide exhibited varying numbers and lengths of neurites, displaying typical neuron-like morphology with round or cone-shaped cell bodies and complex neurite growth. In stark contrast, cells in the control group, TAT-Isl1-ELE-2 group, and TAT-Isl1-ELE-3 group still exhibited astrocyte-like flattened and polygonal morphology. Figure 3 (As shown in Figure 4). Immunofluorescence staining results revealed that, after 28 days of cell treatment, cells treated with the TAT-Isl1-ELE-1 peptide showed positive staining for both the neuronal marker MAP2 and the motor neuron-specific marker ChAT (Figure 4). This result indicates that the TAT-Isl1-ELE-1 peptide can effectively reprogram mouse spinal cord reactive astrocytes into motor neuron-like cells in vitro.

Claims

1. A polypeptide that mimics Isl1, characterized in that, The polypeptide is TAT-Isl1-ELE-1 polypeptide, and its amino acid sequence is GRKKRRQRRRPQGGSGHSGALREDGLFCRADHDVVERASL.

2. The polypeptide according to claim 1, characterized in that, In the amino acid sequence, GRKKRRQRRRPQ is the TAT sequence that promotes the entry of the polypeptide into the cell, GGSGHSG is the linker peptide, and ALREDGLFCRADHDVVERASL is the target sequence, which is amino acid 121-141 of the human Isl1 protein.

3. A pharmaceutical composition, characterized in that, It comprises the polypeptide of claim 1 or 2 and a pharmaceutically acceptable carrier.

4. The pharmaceutical composition according to claim 3, characterized in that, The carrier is an excipient, suspending agent, filler, or diluent.

5. The pharmaceutical composition according to claim 3, characterized in that, The dosage form of the pharmaceutical composition is an injection.

6. The use of the polypeptide of claim 1 or the pharmaceutical composition of claim 3 in inducing cell transformation.

7. The application according to claim 6, characterized in that, The transformation involves a change in cell morphology towards a neuron-like form, followed by reprogramming into neurons.

8. The application according to claim 7, characterized in that, The neurons are MAP2+ mature neurons and ChAT+ motor neurons.

9. The application according to claim 6, characterized in that, The cells in question are reactive astrocytes.

10. The use of the polypeptide of claim 1 in the preparation of a drug for treating SCI.

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